Structural characteristics of two highly selective opioid peptides

The demonstration of opioid receptors by radioligand binding and the discovery of their endogenous peptide ligands has provided a new class of compounds that can be used for the development of novel opioids. The number of potential receptor targets for such opioids has been expanded by the identification of multiple opioid receptor types. The development of highly selective opioid peptides using the principles of conformational restriction permits the analysis of the structure-activity requirements of each receptor type, and is facilitating the elucidation of the functional properties of the different opioid receptors.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP-receptor selective scaffolds. Part II

The nociceptin opioid receptor (NOP) and its endogenous peptide ligand nociceptin/orphanin FQ have been shown to modulate the pharmacological effects of the classical opioid receptor system. Suppression of opioid-induced reward associated with mu-opioid receptor (MOP)-mediated analgesia, without decreasing anti-nociceptive efficacy, can potentially be achieved with NOP agonists having bifunctional agonist activity at MOP, to afford ‘non-addicting’ analgesics. In Part II of this series, we describe a continuing structure–activity relationship (SAR) study of the NOP-selective piperidin-4-yl-1,3-dihydroindol-2-one scaffold, to obtain bifunctional activity at MOP, and a suitable ratio of NOP/MOP agonist activity that produces a non-addicting analgesic profile. The SAR reported here is focused on the influence of various piperidine nitrogen aromatic substituents on the ratio of binding affinity and intrinsic activity at both the NOP and MOP receptors.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I.

The nociceptin receptor (NOP) and its endogenous agonist, nociceptin/orphanin FQ (N/OFQ), members of the opioid receptor and peptide families respectively, modulate the pharmacological effects of classical opioids, particularly opioid-induced reward and nociception. We hypothesized that compounds containing both NOP and opioid receptor activity in a single molecule may have useful pharmacological profiles as non-addicting analgesics or as drug abuse medications. We report here our forays into the structure–activity relationships for discovering ‘bifunctional’ NOP–mu opioid receptor (MOP) ligands, starting from our NOP-selective scaffolds. This initial SAR suggests pharmacophoric elements that may be modified to modulate/increase opioid affinity, while maintaining high affinity for the NOP receptor, to result in potent bifunctional small-molecule NOP/MOP ligands.     

Potential of Nociceptin/Orphanin FQ Peptide Analogs for Drug Development

Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, μ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP-N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide-based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

Bioactive conformations of two seminal delta opioid receptor penta‐peptides inferred from free‐energy profiles

Delta‐opioid (DOP) receptors are members of the G protein‐coupled receptor (GPCR) sub‐family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu‐opioid (MOP), kappa‐opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr‐D ‐Ala‐Gly‐Phe‐D ‐Leu) and DPDPE (Tyr‐D ‐Pen‐Gly‐Phe‐D ‐Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta‐peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over‐simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond‐scale molecular dynamics and bias‐exchange metadynamics simulations. Free‐energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 21–27, 2014.     

Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation

The endoplasmic reticulum (ER) is recognized as an important site for regulating cell surface expression of membrane proteins. We recently reported that only a fraction of newly synthesized delta opioid receptors could leave the ER and reach the cell surface, the rest being degraded by proteasomes. Here, we demonstrate that membrane-permeable opioid ligands facilitate maturation and ER export of the receptor, thus acting as pharmacological chaperones. We propose that these ligands stabilize the newly synthesized receptor in the native or intermediate state of its folding pathway, possibly by inducing stabilizing conformational constrains within the hydrophobic core of the protein. The receptor precursors that are retained in the ER thus represent fully competent folding intermediates that can be targets for pharmacological intervention aimed at regulating receptor expression and cellular responsiveness. The pharmacological chaperone action is independent of the intrinsic signaling efficacy of the ligand, since both agonists and antagonists were found to promote receptor maturation. This novel property of G protein-coupled receptor ligands may have important implications when considering their effects on cellular responsiveness during therapeutic treatments.     

Ligand-directed functional selectivity at the mu opioid receptor revealed by label-free integrative pharmacology on-target

Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR) sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT) approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR) arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs.     

Complementarity of delta opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.     

Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1–1 mg kg⁻¹) and morphine (0.1–10 mg kg⁻¹) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01–0.1 mg kg⁻¹) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.     

Buprenorphine‐elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ‐, μ‐opioid and nociceptin receptors

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ‐ (KOP), μ‐opioid (MOP) and nociceptin/opioid receptor‐like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium‐labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N‐linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U‐69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration‐dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP‐expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration‐dependent AC superactivation elicited by chronic buprenorphine exposure.     

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ₁ or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.     

Molecular determinants of non-specific recognition of delta, mu, and kappa opioid receptors

Identification of the molecular determinants of recognition common to all three opioid receptors embedded in a single three-dimensional (3D) non-specific recognition pharmacophore has been carried out. The working hypothesis that underlies the computational study reported here is that ligands that bind with significant affinity to all three cloned opioid receptors, delta, mu, and kappa, but with different combinations of activation and inhibition properties at these receptors, could be promising behaviorally selective analgesics with diminished side effects. The study presented here represents the first step towards the rational design of such therapeutic agents. The common 3D pharmacophore developed for recognition of delta, mu, and kappa opioid receptors was based on the receptor affinities determined for 23 different opioid ligands that display no specificity for any of the receptor subtypes. The pharmacophore centers identified are a protonated amine, two hydrophobic groups, and the centroid of an aromatic group in a geometric arrangement common to all 23, non-specific, opioid ligands studied. Using this three-dimensional pharmacophore as a query for searching 3D structural databases, novel compounds potentially involved in non-specific recognition of delta, mu, and kappa opioid receptors were retrieved. These compounds can be valuable candidates for novel behaviorally selective analgesics with diminished or no side effects, and thus with potential therapeutic usefulness.     

Design and synthesis of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of GlyT1 inhibitors: achieving selectivity against the mu opioid and nociceptin/orphanin FQ peptide (NOP) receptors

A novel class of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. A novel, straightforward and efficient synthetic strategy for the assembly of the target molecules is also presented.     

Pyrrolo- and pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse.     

High resolution NMR conformational studies of new bivalent NOP receptor antagonists in model membrane systems

The interaction of new bivalent NOP receptor antagonists with dodecyl phosphatidylcholine micelles and DMPC/cholesterol liposomes was investigated in solution by high resolution NMR. The ligands are structurally related to the NOP antagonist JTC-801 plus a propanediamine or heptanediamine spacer between the pharmacophoric units. Ligand internuclear distances were derived from 2D NOESY data and applied to molecular modelling calculations as conformational restraints. NMR experiments on micelles evidenced that the ligands closely approached the micelles but gave no hints on the preferential conformations of the interacting ligands. Results from NMR experiments in the presence of liposomes clearly indicated that both ligands strongly interacted with the bilayer assuming a preferential folded conformation with the quinoline arms superimposing on each other. The finding suggested that these strongly lipophilic pharmacophores could localize in the native receptorial membrane in the form of a depot, gaining access to the recognition site via the lipid bilayer.     

Non-opioid actions of opioid peptides

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.     

Effects on Inflammation of Newly-Synthesized Hexapeptide with Affinity to Opioid and Nociceptin Receptors

Following the discovery of the N/OFQ/NOP system and its modulatory role in physiological and pathophysiological processes, intensive study has started to find selective NOP ligands with hypothetic therapeutic potential. Among the agonists, a hexapeptide Ac-RYYRWK-NH₂ has been identified. It expresses high NOP receptor affinity and selectivity. Its molecule was used as a template, in which Tyr⁵ was substituted by original β²-tryptophan analogue (S)-2-(1-methyl-1H-indol-3-yl)propionic residue (compound HP3) The new compound activates both NOP and opioid receptors. Having in mind that classical opioids, as well as nociceptin, are involved in modulating pain and inflammation, we examined the anti-inflammatory effect of newly-synthesized peptide HP3 on carrageenan-induced peripheral inflammation, and compared it with that of indomethacin (3 mg/kg). It was found that HP3 in dose 40 μg/kg exerts weaker anti-inflammatory action in the first 180 min of the experiment, but is equally effective with indomethacin 3 mg/kg at the end of the observation. The HP3 effect is due mainly of the activation of opioid receptors.     

Semisynthetic neoclerodanes as kappa opioid receptor probes

Modification of the furan ring of salvinorin A (1), the main active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. Conversion of the furan ring to an aldehyde at the C-12 position (5) has allowed for the synthesis of analogues with new carbon-carbon bonds at that position. Previous methods for forming these bonds, such as Grignard and Stille conditions, have met with limited success. We report a palladium catalyzed Liebeskind-Srogl cross-coupling reaction of a thioester and a boronic acid that occurs at neutral pH and ambient temperature to produce ketone analogs at C-12. To the best of our knowledge, this is the first reported usage of the Liebeskind-Srogl reaction to diversify a natural product scaffold. We also describe a one-step protocol for the conversion of 1 to 12-epi-1 (3) through microwave irradiation. Previously, this synthetically challenging process has required multiple steps. Additionally, we report in this study that alkene 9 and aromatic analogues 12, 19, 23, 25, and 26 were discovered to retain affinity and selectivity at kappa opioid receptors (KOP). Finally, we report that the furan-2-yl analog of 1 (31) has similar affinity to 1. Collectively, these findings suggest that different aromatic groups appended directly to the decalin core may be well tolerated by KOP receptors, and may generate further ligands with affinity and activity at KOP receptors.